CN106404879A - Compositions and processes for improved mass spectrometry analysis - Google Patents

Abstract

The invention provides a novel additive for improved analysis by mass spectrometry. More specifically, ascorbic acid has been found to reduce or eliminate the presence of adducts commonly present in mass spectra. The improved processes and compositions of the invention allow for increased accuracy, sensitivity and throughput for samples analyzed by mass spectrometry.

Description

Compositionss for improved mass spectral analyses and method

Present patent application is the invention entitled " group for improved mass spectral analyses that on January 14th, 2009 submits to Compound and method ", national applications number are the PCT state of 200980108864.2 (international application no is PCT/US2009/031020) The divisional application of the National Phase in China of border application.

Related application

The U.S. Non-provisional Patent application that patent application claims were submitted on January 15th, 2008 the 12/014671st Priority, application artificial Thomas Bake that (Thomas Becker) of this non-provisional, invention entitled " use Compositionss and method (COMPOSITIONS AND PROCESSES FOR IMPROVED MASS in improved mass spectral analyses SPECTROMETRY ANALYSIS) ", attorney is SEQ-6015-UT.In the authority allowing, this non-provisional patent The full content of application is incorporated herein by reference, including all words and accompanying drawing.

Invention field

The present invention relates generally to the compositionss for mass spectral analyses and method.

Background

Mass spectral analyses are the very effective analytical tools for the molecular weight of analyte in measuring samples.When using flight During time mass spectrum instrument, the speed of ion flight is about faster by 10 than the speed that molecule migrates in running gel⁷Times, therefore mass spectrum divides Analysis provides a kind of analysis method being exceedingly fast, even mass-spectrometer measurement must repeat 10-100 time to obtain good signal to noise ratio.

Mass spectral analyses generally make sample ionization by various methods at first, for example, pass through matrix assisted laser desorption ionization Or electron spray ionisation (ES) makes sample ionization (MALDI).MALDI prepares and measuring process includes：First by analyte molecule Be embedded in shuttle, be allowed to be in solid liquid is infrared or uv absorption substrate in, described substrate is typically organic acid.Will Comprise substrate and the shuttle of analyte is placed in mass spectrometric ion source.Substrate is made to gasify by short laser pulse, thus Analyte molecule is transferred in gas phase with non-broken state.Analyte molecule is by with the matrix ion collision producing simultaneously and anti- Answer and ionizing.Applied voltage, accelerates ion and enters in field-free flight pipe.Because the quality of ion is different, in ion source from Son is accelerated different speed, and the larger ion of less ion ratio reaches detector earlier.Different flight time conversions For different mass of ions.

It is electron spray ionisation (or ES) that another kind makes the method for analyte ions.Similar with MALDI, electron spray ionisation makes Polar molecule ionization/gasification.First, the sample being analysed to dissolves in a solvent, and in this solvent, sample in a way will Existed with ionized form.In conventional ES method, subsequently make solution by being raised to the thin capillary tube of high potential with pump.? Under atmospheric pressure, less charged drop is sprayed to from ES capillary tube in bath gas, down through pressure and electric potential gradient to Move at the aperture of mass spectrometer high vacuum system.Pass through this path with drop, their desolvated and sizes reduce so that Surface coulomb power has overcome surface tension.Therefore, the drop of breakup of drop Cheng Geng little, the desorbing from drop until ion, or Solvent is completely removed.The exact mechanism that ion is formed also imperfectly understands, but result is to form ion beam, and it is by mass spectrograph Sampling.More detailed descriptions with regard to ES method are shown in what Cole compiled《Electrospray ionization mass spectrometry is analyzed：Ultimate principle, instrument and should With (Electrospray Ionization Mass Spectrometry:Fundamentals,Instrumentation and Applications)(John Wiley and Sons,New York).

No matter using which kind of ionization method, ionization process forms unfavorable adduct can reduce mass spectral analyses and produced The quality of spectrogram and resolution.The detection of analyte and analysis can be led to become tired more specifically, there is unfavorable adduct Difficulty, particularly low abundance or low-quality analyte.

General introduction

The present invention is provided to the method and composition of improved mass spectral analyses.The present invention is provided to the sample of mass spectral analyses Product preparation method, the method is reduced or is farthest reduced the formation of adduct, the party in the way of economic and easy enforcement Formula will not destroy ionization, quality analysiss or the detection of sample ions.The improved method and composition of the present invention is easier to identify With the peak of analyte quantification, thus increasing the number (number of correct calls) of correct calling.These improve channel syndrome Bright to by sodium salt and/or ammonium salt pollution sample and have harmonic analysis thing concentration sample particularly useful.It is described below It is ascorbic acid or its salt, tautomer or the like used in some embodiments, as the interpolation reducing adduct Agent is reducing the presence of adduct in mass spectrum, and improves signal to noise ratio (s/n).Although embodiments of the present invention hereinafter will carry To using " ascorbic acid ", however, it is understood that those of ordinary skill in the art can in method and composition of the present invention Using ascorbic acid, acid ascorbyl ester, its salt, its tautomer or its analog (hereinafter described).

On the one hand, the present invention provides a kind of method, adds in the sample of the analyte comprising stand-by mass spectral analyses for minimizing The formation of compound, the method comprises the following steps：Before analyte being analyzed with mass spectrum, minimizing is added to add in sample The additive of compound, this additive bag contains ascorbic acid.In related embodiment, the additive reducing adduct also can comprise Ammonium oxalate.In some embodiments, the additive of this minimizing adduct reduces adducts with other additive (known or The also undiscovered additive reducing adduct) it is applied in combination.In some embodiments, the additive of this minimizing adduct with One or more resin combination uses.

In some embodiments, analyte is nucleic acid, such as DNA (deoxyribonucleic acid) or ribonucleic acid.Changing described in literary composition Enter the mass spectrometric formats that can be used for producing unfavorable adduct in various operating procedures before mass spectral analyses.Mass spectrometric formats Example includes but is not limited to：Matrix-assisted laser desorption/ionization flight time (MALDI-TOF) mass spectrum (MS), laser desorption matter Spectrum (LDMS), electron spray (ES) mass spectrum, ion cyclotron resonance (ICR) mass spectrum and Fourier transform mass spectrum.Improvement described in literary composition Be readily applied to wherein analyte gasified and ionizing mass spectrometric formats (" MALDI-MS ", such as MALDI-TOF MS, LDMS,ESMS).

The present invention also provides a kind of method, adduct in the sample of the analyte comprising stand-by mass spectral analyses for minimizing Formed, the method comprises the following steps：Before analyte being analyzed with mass spectrum, add in substrate and reduce adduct Additive, this additive bag contains ascorbic acid.The method also includes extra step：With mass spectrum, analyte is being analyzed Before, add the additive reducing adduct in analyte (and substrate).In related embodiment, reduce adding of adduct Plus agent also can comprise ammonium oxalate.In some embodiments, base composition includes 3- hydroxy-picolinic acid (3-HPA), Fructus Citri Limoniae Sour diammonium (DAC) or combinations thereof.In some embodiments, analyte is nucleic acid, such as DNA (deoxyribonucleic acid) or ribose Nucleic acid.In some embodiments, add the additive reducing adduct also in analyte.

In some embodiments, the present invention provides the method that preparation is applied to the base material of mass spectral analyses, and the method includes Following steps：Comprise the host material reducing the additive of adduct, the adding of wherein said minimizing adduct in deposited on substrates Plus agent comprises ascorbic acid.In related embodiment, the additive reducing adduct also can comprise ammonium oxalate.In another correlation In embodiment, the step that the method also includes sealing substrate.The method of sealing substrate includes but is not limited to：By packaging technology The condition realized, such as vacuum sealing and heat seal.In some embodiments, the method is also included with reagent or gas treatment The step farthest to reduce oxidation for the base material.In some embodiments, before sealing, with the indifferent gas of argon etc Body rinses base material.In some embodiments, before with mass spectral analyses, base material is sealed and/or is encapsulated, with limit or It is avoided to be exposed to light or ultraviolet radioactive.In some embodiments, base material is sealed and/or encapsulated, to keep given PH.In some embodiments, base material is encapsulated in a reservoir, described container includes but is not limited to polymer container (for example Polyethylene, polypropylene, polystyrene containers).In some embodiments, base material includes Silicon stone or silicon dioxide.

The present invention also provides the compositionss of stand-by mass spectral analyses, the interpolation that said composition comprises analyte and reduces adduct Agent.In some embodiments, the additive bag reducing adduct contains ascorbic acid.In related embodiment, reduce adduction The additive of thing also can comprise ammonium oxalate.

In some embodiments, the present invention provides and is applied to the compositionss being analyzed with mass spectrum, and said composition comprises Analyte and the additive reducing adduct, described additive bag contains ascorbic acid.Said composition also can comprise ammonium oxalate.

In some embodiments, the present invention is provided to the target position of mass spectral analyses, this target position includes base material and minimizing adds The additive of compound, described additive bag contains ascorbic acid.This target position also can comprise ammonium oxalate.In some embodiments, should Target position also can comprise host material.In related embodiment, host material preload is on base material.In some embodiments In, described target position also can comprise analyte.In some embodiments, described compositionss are sealings.The method of sealing substrate Including but not limited to：Vacuum sealing and heat seal.In some embodiments, with reagent or gas treating composition, with maximum Degree ground reduces oxidation.In some embodiments, before sealing, compositionss are rinsed with the noble gases of argon etc.? In some embodiments, compositionss are sealed and/or is encapsulated, to limit or to avoid it to be exposed to light or ultraviolet radioactive.? In some embodiments, compositionss are sealed and/or is encapsulated, to keep given pH.

The present invention also provides a kind of method preparing the analyte for mass spectral analyses, and the method includes：A () makes to comprise point The solution of analysis thing is contacted with the compositionss comprising ascorbic acid or its salt, tautomer or the like, thus preparing for matter The sample of analysis of spectrum；B () this sample is introduced in mass spectrometer.In some embodiments, described compositionss also comprise grass Sour ammonium.Described analyte is sometimes nucleic acid, including but not limited to DNA (deoxyribonucleic acid), ribonucleic acid and similar material or they Combination.In some embodiments, mass spectral analyses are selected from the group：Matrix-assisted laser desorption/ionization flight time (MALDI- TOF) mass spectrum, laser desorption ionization mass spectrometry (LDMS), electron spray (ES) mass spectrum, ion cyclotron resonance (ICR) mass spectrum and Fourier transform Mass spectrum.In some embodiments, said composition comprises ascorbic acid, and in some embodiments, said composition does not comprise The analog of ascorbic acid.

The present invention also provides a kind of method analyte being analyzed by mass spectrum, and the method includes：A sample is drawn by () Enter in mass spectrometer, described sample comprises analyte and ascorbic acid or its salt, tautomer or the like；B () passes through Mass spectrum is analyzed to sample.In some embodiments, described sample also comprises ammonium oxalate.Described analyte is sometimes core Acid, including but not limited to DNA (deoxyribonucleic acid), ribonucleic acid and similar material or combinations thereof.In some embodiments In, this sample comprises analyte and ascorbic acid, and in some embodiments, this sample does not comprise the similar of ascorbic acid Thing.

The present invention also provides a kind of base material including array of spots (array of spots), and described each speckle includes (i) For the mass spectrographic substrate of matrix-assisted laser desorption/ionization (MALDI) and (ii) ascorbic acid or its salt, tautomer or class Like thing.In some embodiments, each speckle also includes ammonium oxalate, and in some embodiments, one or more speckles Also include analyte.Described analyte is sometimes nucleic acid, including but not limited to DNA (deoxyribonucleic acid), ribonucleic acid and similar thing Matter or combinations thereof.In some embodiments, substrate includes 3- hydroxy-picolinic acid (3-HPA) or other is applied to and passes through The substrate of MALDI analyzing nucleic acids by mass spectrometry.In some embodiments, ascorbic acid and the speckle of substrate (such as 3-HPA) are comprised Compositionss absorb ultraviolet light [for example, absorb about 220nm to about 300nm (e.g., from about 260nm to about 270nm；Purple 266nm) Outer light].In some embodiments, substrate comprises to be applied to the component by MALDI mass spectral analyses protein or peptide, this component Including but not limited to：Ferulic acid, sinapic acid, alpha-cyano -3- Hydroxy-cinnamic acid and alpha-cyano -4- Hydroxy-cinnamic acid.At some In embodiment, base material is chip (such as silicon).In some embodiments, each speckle comprises ascorbic acid, and one In a little embodiments, each speckle does not comprise the analog of ascorbic acid.

Can be included but is not limited to by the improved mass spectrographic example of the method for the present invention and compositionss：Nucleic acid sequencing, gene Typing or methylation analysis.This analysis can be the qualitative or quantitative analysis being carried out by mass spectrum.

Brief Description Of Drawings

Fig. 1 shows using 17 mer synthetic oligo (GTG GTG GTG in standard unmodified substrate for the direct point sample GTG GTG GT) mass spectrum that produces.In the figure, the presence of ammonia adduct is identified that (peak heights are about at 5335Da The 12% of parent peak height).

Fig. 2 shows the identical 17 mer synthetic oligo (GTG using direct point sample on the modified matrix of ascorbic acid GTG GTG GTG GTG GT) mass spectrum that produces.As shown in FIG., no longer there is ammonia adduct.

Fig. 3 shows the mass spectrum that the RhD gene extension product in standard unmodified substrate produces by point sample, obtains 7571Da extends peak and the+55Da adduct peak (NH3+K) in 7626Da.Adduct peak leads to false positive insertion calling (false positive insertion call)(SNR:2；Probability:88%).

Fig. 4 shows the mass spectrum that the RhD gene extension product on the modified matrix of ascorbic acid produces by point sample, obtains The extension product peak of 7571Da, but there is no+55Da adduct peak.False positive insertion calling eliminates (SNR:0；Probability:0%).

Detailed Description Of The Invention

Mass spectrum provides a kind of high precision and sensitive method is used for measuring the molecule of the analyte of nucleic acid and peptide etc Amount.Therefore, mass spectrum provides the very effective instrument that a kind of molecule being difficult to measure to script is analyzed.But, exist Unfavorable addition product can lead to be difficult to accurately detect and analysis of analytes, particularly low abundance or low-quality analyte. When the part as multiple reaction detects multiple analytes in single mass spectrum, problem is more complicated.

Form adduct when ion (typically cation) is associated with biomolecule under the conditions of mass spectral analyses, thus produce The unfavorable mass peak of life.Sample treatment (such as biochemistry, sample operation, sample distribution etc.), sample cleanup (for example, add Resin), any part of sample ionization or sample detection all may promote the formation of adduct.

In the case of matrix-assisted laser desorption/ionization (MALDI) mass spectrum, matrix material itself can be used as formation adduction The source of thing.For example, the mixture of 3- hydroxy-picolinic acid (3-HPA) and dibasic ammonium citrate (DAC) is commonly used for MALDI base nucleic acid Effective ultraviolet substrate of analysis.Add ammonium salt (such as DAC) in substrate (particularly 3-HPA), reason is that people have known For single stranded DNA (ssDNA), these ammonium salts can reduce formation (Wu, J.K. etc., the mass spectrum of cation adduct further in road News flash (Rapid Comm.Mass Spectrom.) 1993,7,191；Pieles etc., nucleic acids research (Nucleic Acid Research),1993,21,14,3191).But, results presented herein shows, DAC is to form ammonia (NH₃) adduct master Want source.For example, Fig. 1 is shown in mass spectrographic+17Da place and there is NH₃Adduct mass peak.

Another kind of material promoting ammonia adduct to be formed is ammonification cation exchange resin, and this resin can be used in MALDI Make before analysis analyte desalination (Nordhoff, E. etc., mass spectrum news flash (Rapid Comm.Mass Spectrom.) 1992,6, 771).Described in following article embodiment, ammonia adduct is mainly formed with guanine and thymus pyrimidine alkali.Therefore, in core When acid analysis thing is rich in these alkali, in analysis, the problem of these adducts is more prominent.

In addition, ammonification cation exchange resin can not completely remove the whole sodium ion in DNA backbone.This removal is not Fully lead to that sodium ion adduct mass peak occurs at+22Da.Other adducts can be formed by host material, there are thymus During pyrimidine base more commonly.For example, when being used 3- hydroxy-picolinic acid as substrate, find adduction at 94,138 and 188Da Amount of substance peak.All these adduct summits integrally lead to the explanation of error to mass spectral results.Therefore, greatly reduce adduct The amount of (particularly alkaline adduct and ammonia adduct) and the compositionss of frequency and method can be used for improving the accurate of mass spectral analyses Degree, sensitivity and analysis throughput.The additive that there is minimizing adduct can reduce or eliminate the interpolation not using minimizing adduct Adduct peak present in the sample analyzed during agent.As described herein, when extension product intensity is suitable, with resin treatment Standard substrates are compared, and add ascorbic acid average adduct can be formed fraction (formation score) (defined in literary composition ) at most reduce by 40% (referring to embodiment 1-3).In addition, with the additive agent modified substrate of ascorbic acid verified up to 6 Physics and chemically stable (referring to embodiment 4) is kept, this allows for use Vitamin C during manufacturing in the time of the moon Low-kappa number base material.

Analyte

The present invention can improve the mass spectral analyses of analysis below thing：For example, nucleic acid (as oligonucleotide and polynucleotide), egg White matter, peptide and lipid, including their particular analog and conjugate, such as glycoprotein or lipoprotein.It is applied to institute of the present invention The other materials stating maldi analysis are small molecule, metabolite, natural prodcuts and medicine.The method of the present invention and compositionss are special It is applied to the polynucleotide mainly comprising guanine and thymus pyrimidine, because these polynucleotide are more readily formed ammonia adduct.

In literary composition, term " nucleic acid " used refers to single-stranded and/or double-stranded polynucleotide, such as DNA (deoxyribonucleic acid) (DNA) and core Ribosomal ribonucleic acid (RNA), and the analog of RNA or DNA or derivant.Term " nucleic acid " also includes the analog of nucleic acid, such as peptide Nucleic acid (PNA), phosphorothioate dna, acyclic nucleoside and other this analogs and derivatives or combinations thereof.

The nucleotide analog comprising in polynucleotide can be the nucleotide that for example quality changes, so that polynucleotide Quality variant；Nucleotide containing detectable label, described labelling is, for example, fluorescent labeling, radioactive label, luminous mark Note or chemiluminescent labeling, thus polynucleotide can be detected；Or the nucleotide containing active group, described active group is, for example, Biotin or thiol group, contribute to fixing on a solid support polynucleotide.Polynucleotide also can comprise one or more The main chain key of alternative cracking, for example, chemical cracking, enzymatic lysises or photodestruciton.For example, polynucleotide may include one or many Individual deoxyribonucleotide, is followed by one or more ribonucleotides, followed by one or more deoxyribonucleotides, This sequence can be cracked at ribonucleotide by basic hydrolysiss.It is resistance to that polynucleotide may also include one or more comparisons The key of cracking, for example, chimeric oligonucleotide primer, this chimeric oligonucleotide primer may include the nucleoside bonded by peptide nucleic acid(PNA) Acid with least one at 3' end by phosphodiester bond or similar bonded nucleotide, it can pass through polymerase extension.

Sample

Used in literary composition, " sample " refers to the compositionss containing analyte to be analyzed.In some embodiments, sample comes From " biological sample ".Biomaterial is typically considered by (for example, people, animal, plant, antibacterial, funguses, the primary life of live body source Thing, virus) any material that obtains.Biological sample can be any form, including solid material (such as tissue, cell granule And living tissue) and biofluid [such as urine, blood, saliva, amniotic fluid and collutory (containing Stomatocyte)].Preferably solid Material and liquid mixing.

Substrate

Host material is used in some form of mass spectral analyses, such as MALDI mass spectrum.Host material is used for dividing analyte Son is separated from each other and comes, and absorbs the energy that laser photon gives, and by energy transfer to analyte molecule, so that they solve Inhale and ionize.Once analyte is ionized, ion matter can be measured using the mass spectrograph of flight time (TOF) analyser etc Amount.

For the host material of mass spectral analyses selection generally depend on analysis biomolecule type.For example, use matter During analysis of spectrum nucleic acid, conventional host material is 3- hydroxy-picolinic acid (3-HPA) and the mixture of dibasic ammonium citrate (DAC). Another kind of host material for promoting sample analytes ionization is 2,5- resorcylic acid (DHB).DHB also runs into formation and adds Compound and the problem producing the chemical noises that disturbed specimen is analyzed.Alpha-cyano -4- hydroxycinnamic acid (α-CHCA) is in matrix It is widely used in the example of the substrate of ionization protein and peptide analysis thing in auxiliary laser flying time mass spectrum analysis.But, usually Produce α-CHCA adduct, the low abundance of accurate detection, the ability of low quality analyte can be disturbed.Can be with the free radical described in literary composition Scavenger is used for other substrate of the mass spectral analyses of improvement together referring to (the Rapid Comm.Mass Spectrom.12 such as Li: 993-998 (1998), M.C.Fitzgeraldand L.M.Smith (Annu.Rev.Biophys.Biomol.Struc.1995.24:117-40), and (the Mass Spectrometry such as Nordhoff Reviews,1996,15,67-138；All documents are all incorporated herein by reference), the example of substrate includes but is not limited to：2, 4,6- trihydroxy-acetophenone (THAP), ortho-aminobenzoic acid, nicotinic acid, salicylamide, 1- isoquinolin alcohol, T-2- (3- (the 4- tert-butyl group Phenyl) -2- methyl -2- allylidene) Cyanoacetyl-Cyacetazid (DCTB), sinapic acid (SA), dithranol (DIT), 3- quinolin-2-ylamine, trans - 3- indole acrylic acid (IAA), 2- (4- hydroxyphenyl azo) benzoic acid (HABA), succinic acid, 2,6- resacetophenone, Resina Ferulae Acid, caffeic acid, glycerol and nitroaniline.

Before or after by apposition to base material, can be by host material and one or more additive or analyte Combination.When analyte is embedded in light absorbent substrate, this substrate is generally existed with the amount excessive with respect to analyte.For For MALDI method, analyte molecule is attached in conventional crystalline host material during crystallization with some form, or At least being attached to is favourable in the interface between little host crystal.In some embodiments, first by additive and substrate Material mixes in the solution, the host material/additive solution of merging is deposited on base material, crystallizes on the substrate.

In various embodiments, discrete spot will be formed on apposition to base material by following steps：By substrate It is dissolved in the solution comprising ascorbic acid and suitable solvent such as water.Resulting solution is deposited on MALDI base material, by gained Base material is put in a vacuum chamber, so that substrate/ascorbic acid solution is dried under vacuum.In one embodiment, ascorbic acid list Solely or with other substrate combination it is used as host material.

Using various methods, substrate, additive or analyte can be deposited on base material.In one embodiment, in list Apply each composition in only step.For example, it is possible to by host material preload on base material, then be distributed using suitable liquid Equipment [such as piezoelectricity, pin type feed head (pin tool) distributor] adds analyte.In some embodiments, described all Composition deposits in combination.For example, first substrate and additive can be combined (dissolving in a solvent), deposit to together On base material, then add analyte.In some embodiments, substrate or substrate/additive deposit can be dried on base material, Evaporate with solvent and form host crystal.Then in the substrate deposited atop analyte solution being dried, make the apposition of drying Thing is partly dissolved and the substrate that again dissolves and analyte cocrystallization.

In some embodiments, the additive reducing adduct is directly used as host material, and in some embodiments In, the additive reducing adduct is used as the component of host material.In some embodiments, host material includes reducing adduction Mass spectrometer matrix material described in the additive of thing and one or more literary composition (such as 3-HPA, DAC, DHB, CHCA, THAP, One or more of DCTB, DIT, SA, IAA, HABA).For the additive reducing adduct and one or more mass spectrum base The embodiment that material is used together, the content range reducing the additive of adduct is 99 weights accounting for host material gross weight Amount % to 1 weight % (for example, account for about 5 weight % of host material gross weight, 10 weight %, 15 weight %, 20 weight %, 25 Weight %, 30 weight %, 35 weight %, 40 weight %, 45 weight %, 50 weight %, 55 weight %, 60 weight %, 65 weights Amount %, 70 weight %, 75 weight %, 80 weight %, 85 weight %, 90 weight % or 95 weight %), sometimes reduce adduct The mol ratio (i.e. the ratio of the molal quantity of the molal quantity of additive and mass spectrometer matrix) e.g., about 1 of additive:20、1:19、1: 18、1:17、1:16、1:15、1:14、1:13、1:12、1:11、1:10、1:9、1:8、1:7、1:6、1:5、1:4、1:3 or 1:2.

Additive

In literary composition, term used " reducing the additive of adduct " is incorporated into needed for any one or more of mass spectral analyses Component or reagent in material.These components or reagent include sample, analyte, host material, base material or combinations thereof. In some embodiments, the additive reducing adduct is ascorbic acid, or it has essentially identical minimizing adduct effect Any derivant of fruit.

In some embodiments, the additive reducing adduct is free radical scavenger.Can be applied to using any Mass spectral analyses, are applied to the free radical scavenger of the mass spectral analyses of nucleic acid in some cases.The example bag of free radical scavenger Include but be not limited to：Ascorbic acid, retinal, tocotrienol, tocopherol, coenzyme Q10, melatonin, lycopene, leaf are yellow Element, alpha-carotene, beta-carotene, cryptoxanthin, astaxanthin, canthaxanthin, flavone (such as digicitrine, 4',5,7-trihydroxyflavone, mandarin orange Flavone), flavonol (favonols) (such as Quercetin, kaempferol, myricetin, isorhamnetin, proanthocyanidin), flavanone (favanones) (such as hesperetin (hasperetin), naringenin, eriodictyol), isoflavone phytoestrogens (such as dyewood Flavone, Daidezin, Glycitein), class compound (stilbenoids) (such as resveratrol, Lignum pterocarpi indici), anthocyanin (such as cyanide (cyaniding), delphinidin, malvidin, pelargonidin, peonidin, petunidin), phenolic acid and ester (for example ellagic acid, gallic acid, salicylic acid, rosmarinic acid, cinnamic acid, chlorogenic acid, chicoric acid, gallotannin, tan spend tan Matter), non-flavonoid phenolic compound (nonfalvonoid phenolics) (such as curcumin, ton ketone, silymarin, fourth Eugenol) and organic oxidation-resistant agent (such as citric acid, oxalic acid, phytic acid, lignan, uric acid, N-acetylcystein).This area Those of ordinary skill be easy to by parallel parsing (side-by-side analyses) as be shown in the examples to this A little scavengers carry out conventionally test to identify the free radical scavenger being suitable as mass spectrographic additive or substrate.

In some embodiments, additive is substantially free of impurity, does not therefore need to carry out purification.If minimizing adduct Additive purity not, can be logical by this additive of method purification as known in the art, to remove impurity, for example Cross ion-exchange resin purification and carry out this purification process.

Additive can dissolve (for example, being dissolved in water) in liquid form, is subsequently deposited upon in the substrate of preload, or It is deposited directly on base material in the case of no preload substrate.Or, additive can be mixed with substrate, be subsequently deposited upon On base material.Additive can be mixed with substrate, obtain 1 and arrive the substrate concentration of about 20mg/ml and the additive concentration of about 5-50mM. If the consumption of additive is not enough it is impossible to significantly reduce the formation of adduct, and may suppress matter using excessive additive The parent signal of spectrum.Those skilled in the art need not be excessive experiment just by changing the amount of additive and it can be judged to spectrogram spy The impact levied determining the appropriate amount of additive, to optimize the analysis of specific analyte.

In some embodiments, the additive reducing adduct can be used alone, or be reduced or avoided unfavorable plus The other materials that compound exists are applied in combination.Ascorbic acid additive can be with the other additive groups that can reduce mass spectrum background noise Close and use.Other known appropriate addn includes resin, volatility ammonium salt, particularly volatility list alkali formula (monobasic), Two alkali formulas (dibasic) or three alkali formula (tribasic) ammonium salts.The alkalescence of salt additives should not be too high, in order to avoid interference is analyzed Sample.Additive can be single subphosphate and sulfate (for example single alkali formula ammonium phosphate) and two alkali formula citrates are (for example Two alkali formula ammonium citrates) and three alkali formula citrates (such as three alkali formula ammonium citrates).

In some embodiments, the additive reducing adduct is ascorbic acid, acid ascorbyl ester, its salt, its change Isomer or the ascorbic acid analogs (including analog salt and tautomer) with formula：

Wherein：

R¹And R²It is independently OH, halogen, R³、OR³, azido, cyano group, CH₂R³、CHR³R⁴、SR³、NR³R⁴；

R³And R⁴It is independently H, alkyl, acetenyl or cyano group, or optionally substituted aryl carbocyclic ring, aryl-heterocyclic, non-aryl Carbocyclic ring or nonarylheterocyclic.Used in literary composition, ascorbic acid analogs are typically free radical scavenger, ordinary skill people Member can determine that ascorbic acid free radical scavenging activity (for example, with oxidant such as 2,6- chlorophenesic acid-indophenols (DCPIP), iodine, Iodate and iodine compound mixture or N-bromosuccinimide are titrated).

Used in literary composition, term is " optionally substituted " represents that described special groups or group group can not have non-hydrogen to replace Base, or described special groups or group group can have one or more non-hydrogen substituent.Unless otherwise stated, there may be The sum of substituent group is equal to the H atom number present on described group of unsubstituted form.If optional substituent group passes through double bond Connect, such as ketonic oxygen (=O), then this group occupies two available prices, so according to the number that can utilize price, can wrap The total number of the substituent group containing reduces.

Ascorbic acid and the like can have the group of energy ionizing, so can be obtained as salt.In this case, no By when mentioning compound, in the art all it should be understood that pharmaceutically acceptable salt can also be used.These salt can be related to And inorganic or organic acid acid-addition salts, or prepared by inorganic or organic base when the compound of the present invention is sour form Salt.Generally, compound is made or be used as pharmaceutically acceptable salt, this pharmaceutically acceptable salt is as pharmaceutically acceptable Acid or alkali addition compound product be obtained.Suitably pharmaceutically acceptable bronsted lowry acids and bases bronsted lowry is well known in the art, for example For forming hydrochloric acid, sulphuric acid, hydrobromic acid, acetic acid, lactic acid, citric acid or the tartaric acid of acid-addition salts, for forming basic salt Potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amine etc..Fully establish preparation in the art suitable The method of salt.In some cases, compound can contain acid functional group and alkali functional group simultaneously, and they can have in this case Two ionogens, but no net charge.

Ascorbic acid analogs usually contain one or more chiral centres.The present invention includes various independent stereoisomerisms Form and the stereoisomer mixture of different chiral purity, including racemic mixture.Also include being formed is various non- Enantiomer and tautomer.The compound of the present invention can be presented in more than one tautomer；Herein Described in be used for the purpose of for the sake of simplicity it is thus understood that including other tautomeric forms for a kind of tautomer.

Term " alkyl ", " thiazolinyl " and " alkynyl " inclusion straight chain used in literary composition, side chain and cyclic monovalent hydrocarbon and it Combination, only contain only C and H when they are unsubstituted.Example includes methyl, ethyl, isobutyl group, cyclohexyl, cyclopenta Ethyl, 2- acrylic, 3- butynyl etc..Sometimes the total number of carbon atoms of each group is described, such as when group can at most contain in literary composition During 10 carbon atoms, it is represented by 1-10C or C1-C10 or C1-10.Replace carbon former when allowing hetero atom (usually N, O and S) The period of the day from 11 p.m. to 1 a.m, such as, in miscellaneous alkyl, although the number of description group is still written as such as C1-C6, it represents carbon atom in group Number and the heteroatomic number sum replacing carbon atom in described ring or chain backbone.

Generally, the alkyl of the present invention, thiazolinyl and alkynyl substituted base can contain a 10C (alkyl) or two 10C (thiazolinyl or Alkynyl).It is preferred that they contain a 8C (alkyl) or two 8C (alkenyl or alkynyl).Sometimes they contain a 4C (alkane Base) or two 4C (alkenyl or alkynyl).An independent group may include the multiple bond of more than one type, or more than one is multiple Build；When these groups contain at least one carbon-carbon double bond, they include in the definition of term " thiazolinyl ", when these groups contain When having at least one triple carbon-carbon bonds, they are included in the definition of term " alkynyl ".

Alkyl, thiazolinyl and alkynyl are generally optionally substituted to a certain extent, as long as these replacements are had a mind in chemistry Justice.Typical substituent group includes but is not limited to halogen ,=O ,=N-CN ,=N-OR ,=NR, OR, NR₂、SR、SO₂R、 SO₂NR₂、NRSO₂R、NRCONR₂、NRCOOR、NRCOR、CN、COOR、CONR₂, OOCR, COR and NO₂, wherein each R is independently H, C1-C8 alkyl, C2-C8 miscellaneous alkyl, C1-C8 acyl group, the miscellaneous acyl group of C2-C8, C2-C8 thiazolinyl, C2-C8 miscellaneous thiazolinyl, C2-C8 alkynes The miscellaneous alkynyl of base, C2-C8, C6-C10 aryl or C5-C10 heteroaryl, each R is optionally by following substituent group：Halogen ,=O ,=N- CN ,=N-OR ' ,=NR ', OR ', NR '₂、SR’、SO₂R’、SO₂NR’₂、NR’SO₂R’、NR’CONR’₂、NR’COOR’、NR’ COR’、CN、COOR’、CONR’₂, OOCR ', COR ' and NO₂, wherein each R ' be independently H, C1-C8 alkyl, C2-C8 miscellaneous alkyl, The miscellaneous acyl group of C1-C8 acyl group, C2-C8, C6-C10 aryl or C5-C10 heteroaryl.Alkyl, thiazolinyl and alkynyl also can be by C1-C8 acyls The miscellaneous acyl group of base, C2-C8, C6-C10 aryl or C5-C10 heteroaryl replace, and these substituent groups each can be by suitable concrete group Substituent group replace.

" acetylene " substituent group is optionally substituted 2-10C alkynyl, and formula is-C ≡ C-Ra, and wherein Ra is H or C1-C8 alkane Base, C2-C8 miscellaneous alkyl, C2-C8 thiazolinyl, C2-C8 miscellaneous thiazolinyl, C2-C8 alkynyl, the miscellaneous alkynyl of C2-C8, C1-C8 acyl group, C2-C8 are miscellaneous Acyl group, C6-C10 aryl, C5-C10 heteroaryl, C7-C12 aryl alkyl or C6-C12 heteroaryl alkyl, each Ra base optionally by One or more substituent groups being selected from the group replace：Halogen ,=O ,=N-CN ,=N-OR ' ,=NR ', OR ', NR ' 2, SR ', SO₂R’、SO₂NR’₂、NR’SO₂R’、NR’CONR’₂、NR’COOR’、NR’COR’、CN、COOR’、CONR’₂, OOCR ', COR ' and NO₂, wherein each R ' be independently H, C1-C6 alkyl, C2-C6 miscellaneous alkyl, C1-C6 acyl group, the miscellaneous acyl group of C2-C6, C6-C10 aryl, C5-C10 heteroaryl, C7-12 aryl alkyl or C6-12 heteroaryl alkyl, these substituent groups are each optionally one or more The substituent group being selected from the group：Halogen, C1-C4 alkyl, C1-C4 miscellaneous alkyl, C1-C6 acyl group, the miscellaneous acyl group of C1-C6, hydroxyl, amino With=O；Two of which R ' formation 3-7 yuan of rings can be connected, this ring optionally contains up to three hetero atoms being selected from N, O and S.? In some embodiments, the Ra in-C ≡ C-Ra is H or Me.

" miscellaneous alkyl ", " miscellaneous thiazolinyl " are similar with corresponding alkyl (alkyl, thiazolinyl and alkynyl) with the definition of " miscellaneous alkynyl " etc., But ' miscellaneous ' represents that this group contains 1-3 O, S or N hetero atom or combinations thereof in their backbone residue；So phase One of at least one of the alkyl answered, alkenyl or alkynyl appointed hetero atom of carbon atom replaces, and forms miscellaneous alkyl, miscellaneous thiazolinyl Or miscellaneous alkynyl.Alkyl, thiazolinyl are generally identical with corresponding alkyl with the size typically and preferably of the hydridization form of alkynyl, exist In the pro forma substituent group of hydridization with identical described in alkyl.Consideration for chemical stability it is to be understood that unless otherwise Illustrate, these groups do not include the adjacent hetero atom of two or more, unless oxo base, such as nitro or sulphonyl existed on N or S Base.

Although " alkyl " described in literary composition includes cycloalkyl and cycloalkyl-alkyl, Wen Zhongke uses term " cycloalkyl " to describe The carbocyclic nonaromatic base being connected by ring carbon atom, can use " cycloalkyl-alkyl " description to be connected with molecule by alkyl linker Carbocyclic nonaromatic base.Similarly, " heterocyclic radical " description can be used as annular atom and to pass through ring containing at least one hetero atom The nonaromatic cyclic group that atom (can be C or N) is connected with molecule；Can use " cycloheteroalkylalkyl " description by linker with another This group that individual molecule connects.It is applied to size and the substituent group of cycloalkyl, cycloalkyl-alkyl, heterocyclic radical and cycloheteroalkylalkyl With identical described in alkyl.Used in literary composition, these terms also include the ring containing one or two double bond, as long as this ring is Non-aromatic.

Used in literary composition, " acyl group " includes such class group, and this group comprises alkyl, thiazolinyl, alkynyl, aryl or alkyl Aryl, they are connected on one of two available prices of carbonylic carbon atom, and miscellaneous acyl group refers to outside carbonyl carbon at least one Individual carbon atom is selected from the corresponding group that the hetero atom of N, O and S replaces.Therefore, miscellaneous acyl group includes such as-C (=O) OR and C (=O) NR₂, and C (=O)-heteroaryl.

Acyl group and miscellaneous acyl group are connected with any group or molecule by the open price of carbonylic carbon atom.Generally, they are C1-C8 acyl group, including formoxyl, acetyl group, valeryl and benzoyl, the miscellaneous acyl group of C2-C8, including Methoxyacetyl, Ethoxy carbonyl and 4- pyridine acyl.Acyl group can be comprised or the alkyl of this kind of group, aryl and the hetero atom of miscellaneous acyl group can be gone up The substituent group of the substituent group of each appropriate section being generally suitable as acyl group or miscellaneous acyl group described in literary composition replaces.

" aromatics " part or " aryl " partly refer to there is the monocyclic of well-known armaticity or fused bicyclic moiety；Example Including phenyl and naphthyl.Similarly, " heteroaromatic " and " heteroaryl " refers to make containing one or more hetero atoms selected from O, S and N This kind of monocyclic or fused bicyclic carbocycle for annular atom.Comprise hetero atom and can keep 5 yuan of rings and the armaticity of 6 yuan of rings.Typical case Heteroaromatic system include monocyclic C5-C6 aromatic group, such as pyridine radicals, pyrimidine radicals, pyrazinyl, thienyl, furyl, pyrroles Base, pyrazolyl, thiazolyl, oxazolyl and imidazole radicals, monocyclic with phenyl ring or any heteroaromatic by one of these monocyclic bases Base condenses the fused bicyclic moiety of formation, forms C8-C10 bicyclic group, such as indyl, benzimidazolyl, indazolyl, benzo three Oxazolyl, isoquinolyl, quinolyl, benzothiazolyl, benzofuranyl, Pyrazolopyridine base, quinazolyl, quinoxalinyl, scold Quinoline base etc..Consider there is any monocyclic of armaticity from the electronics distribution of whole member ring systems or fused bicyclic carbocycle is included in this In definition.The ring also including at least being connected directly between on molecule nubbin in this definition has the bicyclic radicals of armaticity.Logical Often, member ring systems contain 5-12 annular atom.Preferably bicyclic heteroaryl contains 5-6 annular atom, and bicyclic heteroaryl contains 8-10 Annular atom.

Aryl and heteroaryl moieties can be replaced by various substituent groups, these substituent groups include C1-C8 alkyl, C2-C8 thiazolinyl, C2-C8 alkynyl, C5-C12 aryl, C1-C8 acyl group and their hydridization form, these substituent groups itself can be substituted further； Other substituent groups for aryl and heteroaryl moieties include halogen, OR, NR₂、SR、SO₂R、SO₂NR₂、NRSO₂R、NRCONR₂、 NRCOOR、NRCOR、CN、COOR、CONR₂, OOCR, COR and NO₂, wherein each R is independently H, C1-C8 alkyl, the miscellaneous alkane of C2-C8 Base, C2-C8 thiazolinyl, C2-C8 miscellaneous thiazolinyl, C2-C8 alkynyl, the miscellaneous alkynyl of C2-C8, C6-C10 aryl, C5-C10 heteroaryl, C7-C12 Aryl alkyl or C6-C12 heteroaryl alkyl, each R is optionally as the mode described in alkyl above is substituted.Aryl or heteroaryl Substituent group on base can be suitable for each type of substituent group or suitable in these substituent groups by the above certainly further The substituent group of each component of substituent group.Thus, for example, arylalkyl substitutents can be by above institute on aryl moiety The substituent group being generally used for aryl stated replaces, it can further on moieties by the above usual or suitable Substituent group for alkyl replaces.

Similarly, " aryl alkyl " and " heteroaryl alkyl " refer to the aromatics being connected with their junction point by linker and Heteroaromatic ring system, described linker is, for example, alkylidene, including substituted or unsubstituted, saturation or undersaturated, have ring or Acyclic linker.Typical linker is C1-C8 alkyl or their hydridization form.These linkers may also include carbonyl, Them are made to be provided that the substituent group as acyl group or miscellaneous acyl moiety.Aromatic ring in aryl alkyl or heteroaryl alkyl or heteroaryl Ring can be replaced by the above substituent group being equally used for aryl.It is preferred that aryl alkyl include optionally defined above The phenyl ring of the substituent group for aryl and C1- that is unsubstituted or being replaced by one or two C1-C4 alkyl or miscellaneous alkyl C4 alkylidene, wherein alkyl or miscellaneous alkyl are optionally cyclized the ring forming cyclopropane, dioxolanes or tetrahydrofuran etc. Similarly, heteroaryl alkyl preferably includes optionally by the C5- of the substituent group frequently as the substituent group on aryl mentioned above C6 bicyclic heteroaryl and C1-C4 alkylidene that is unsubstituted or being replaced by one or two C1-C4 alkyl or miscellaneous alkyl, or miscellaneous Aryl alkyl includes optionally substituted phenyl ring or C5-C6 bicyclic heteroaryl and unsubstituted or by one or two C1-C4 alkyl Or the C1-C4 miscellaneous alkylidene that miscellaneous alkyl replaces, wherein alkyl or miscellaneous alkyl be optionally cyclized formation cyclopropane, dioxolanes or The ring of tetrahydrofuran etc.

When describing aryl alkyl or heteroaryl alkyl is optionally substituted, substituent group can be in the alkyl of group or miscellaneous alkyl Partly go up or on aryl or heteroaryl moieties.It is optionally present in alkyl or substituent group that miscellaneous alkyl partly goes up and above institute State be usually used in alkyl substituent group identical；The substituent group being optionally present on aryl or heteroaryl moieties is conventional with the above Identical in the substituent group of aryl.

Used in literary composition, " aryl alkyl ", if unsubstituted, is alkyl, with ring and alkylidene or similar linker The total number of carbon atoms be described.Therefore, benzyl is C7- aryl alkyl, and phenylethyl is C8- aryl alkyl.

" heteroaryl alkyl " mentioned above refers to comprise the part of aryl connecting by linker, with " aryl alkyl " Difference is that at least one annular atom of aryl moiety or an atom of linker are selected from N, the hetero atom of O and S.Herein The middle total atom number according to ring and linker describes heteroaryl alkyl, and they include the aryl connecting by miscellaneous alkyl linker； The heteroaryl being connected by alkyl linker (such as alkylidene)；The heteroaryl being connected by miscellaneous alkyl linker.Thus, for example C7- heteroaryl alkyl includes pyridylmethyl, phenoxy group and N- pyrrolylmethoxy.

Used in literary composition, " alkylidene " refers to bivalent hydrocarbon radical；Because this group is bivalence, it can be by two other Group links together.Generally, alkylidene refers to (CH₂)_n-, wherein n is 1-8, and preferably n is 1-4, but in the feelings particularly pointing out In condition, alkylidene can also be not necessarily required to the two of chain for other length, open price by other substituent groups End.Therefore, CH (Me)-and C (Me)₂- it is referred to as alkylidene, ring group such as ring propyl- 1,1- diyl is referred to as alkylene Base.When alkylidene is substituted, substituent group includes the substituent group being normally present on alkyl described in literary composition.

Generally, any alkyl, thiazolinyl, alkynyl, acyl group, aryl or the aryl alkyl that comprise in substituent group or these groups One of any hydridization form itself optionally replaced by other substituent groups.Without other, these substituent groups are described, then The property of these substituent groups is similar to the property described in just primary substituent group.Therefore, in such as R7 be alkyl embodiment in, This alkyl remaining substituent group optionally listed by the embodiment as R7 replaces, as long as this replacement is in chemistry Significant, and this replacement will not destroy the size limitation of alkyl itself；For example, the alkyl being replaced by alkyl or alkenyl is only It is the carbon number upper limit extending these embodiments, it is not included.But, by aryl, amino, alkoxyl ,=O etc. The alkyl replacing is included within the scope of the invention, and the atom of these substituent groups is not counted in for describing described alkyl, thiazolinyl etc. The atomic number of group.When not specifying substituent group number, available according to these alkyl, thiazolinyl, alkynyl, acyl group or aryl Price, they can be replaced by multiple substituent groups；Especially, for example, these groups can be in any or all of available valency Position is replaced by fluorine atoms.

Used in literary composition, " hydridization form " refers to the derivant of the group of alkyl, aryl or acyl group etc, wherein specified Carbocylic radical at least one carbon atom be selected from N, O and S hetero atom replace.Therefore, alkyl, thiazolinyl, alkynyl, acyl The hydridization form of base, aryl and aryl alkyl is miscellaneous alkyl, miscellaneous thiazolinyl, miscellaneous alkynyl, miscellaneous acyl group, heteroaryl and heteroaryl alkane respectively Base.It should be understood that typically not greater than two N, O or S are continuously coupled, unless oxo base is connected on N or S forms nitro or sulphur Acyl group.

Used in literary composition, " halogen " includes fluorine, chlorine, bromine and iodine.It is usually preferable that fluorine and chlorine.Used in literary composition " amino " Refer to NH₂, but when amino is described as " substituted " or " optionally substituted ", this term includes NR ' R ", wherein R ' and R " each From being independently H, or the hydridization form of alkyl, thiazolinyl, alkynyl, acyl group, aryl or one of aryl alkyl or these groups, The hydridization form of alkyl, thiazolinyl, alkynyl, acyl group, aryl or one of aryl alkyl or these groups is all optionally described in literary composition The substituent group being suitable for corresponding group replaces.This term also includes wherein R ' and R " it is joined together to form the form of 3-8 yuan of rings, institute Stating 3-8 yuan of rings can be saturation, undersaturated or armaticity, and it comprises the 1-3 hetero atom independently selected from N, O and S As annular atom, the substituent group that this ring is optionally suitable for alkyl described in literary composition replaces, or if NR ' R " be aryl, then should The substituent group that ring is optionally usually used in heteroaryl described in literary composition replaces.

Used in literary composition, term " carbocyclic ring " refers to comprise only the cycle compound of carbon atom in ring, and " heterocycle " refer to comprise miscellaneous former The cycle compound of son.Carbocyclic ring and heterocycle structure include thering is monocyclic, bicyclic or polycyclic system compound.Term used in literary composition " hetero atom " refers to any atom beyond carbon or hydrogen, such as nitrogen, oxygen or sulfur.The illustrative example of heterocycle includes but is not limited to tetrahydrochysene Furan, 1,3- dioxolanes, 2,3 dihydro furan, pyrans, Pentamethylene oxide., benzofuran, isobenzofuran, the different benzene of 1,3- dihydro And furan, isoxazole, 4,5- dihydro isoxazole, piperidines, pyrrolidine, pyrrolidin-2-one, pyrroles, pyridine, pyrimidine, octahydro pyrroles And [3,4b] pyridine, piperazine, pyrazine, morpholine, thiomorpholine, imidazoles, imidazolidine -2,4- diketone, 1,3- dihydrobenzo imidazoles -2- Ketone, indole, thiazole, benzothiazole, thiadiazoles, thiophene, Tetramethylene sulfide -1,1- dioxide, phenodiazine, triazole, guanidine, diaza Bicyclic [2.2.1] heptane, 2,5- diazabicyclo [2.2.1] heptane, 2,3,4,4a, 9,9a- hexahydro -1H- B-carboline, epoxy second Alkane, expoxy propane, Pentamethylene oxide., dioxane, lactone, aziridine, azetidine, piperidines, lactams are it is also possible to include heteroaryl. The illustrative example of other heteroaryls includes but is not limited to furan, pyrroles, pyridine, pyrimidine, imidazoles, benzimidazole and triazole.

Base material

Used in literary composition, " base material " refers to insoluble carrier, on this carrier sedimentation analysiss thing and being analyzed.Base material can Including but not limited to Silicon stone, glass (such as glass, control hole glass (controlled-pore glass, CPG)), nylon, Wang Shi Resin (Wang resin), Merrifield resin (Merrifield resin), polydextran gel (Sephadex), agarose Gel (Sepharose), cellulose, magnetic bead, Dai Nazhu (Dynabeads), metal surface (for example steel, gold, silver, aluminum, silicon and Copper), plastic material (such as polyethylene, polypropylene, polyamide, polyester, polyvinylidene fluoride (PVDF)) or contact pin (pin) (example As it is adaptable to the pin array of combinatorial compound or analysis, or the beadlet in flat surfaces depression, flat surfaces e.g. have or There is no the chip (such as silicon wafer) of plate.Solid carrier can be any required form, including but not limited to：Beadlet, core Piece, capillary tube, plate, barrier film, chip, rake, contact pin, the chip with contact pin, the surface of substantially flat, depression or nanoliter well The array of (nanoliter wells), and other geometry well known by persons skilled in the art and form.Preferably carrier is It is designed for accepting in discontinuous position or connect the flat surfaces of sample.Most preferably there is the flat table of hydrophobic region Face, described hydrophobic region surrounds the hydrophilic position for accepting, accommodating or combine sample.Base material is to the device using in processes Or the operation of reagent can be inert, including being usually used in the mass spectrographic host material of MALDI and solvent.

On base material, the speckle of substrate or substrate/additive or substrate/analyte/additive generally has certain spy Levy.On base material, the diameter of each speckle may be about 200 μm and arrives 1mm.Spot diameter is typically substantially homogeneous, and speckle is to speckle Diameter change generally should minimize (for example, change is about 20 μm).Can be used for the center of speckle on the base material of mass spectral analyses with The distance between center can be any value, and such as the distance between Center-to-Center is 2.25mm or 1.125mm, for example, base On material, the distance between Center-to-Center of speckle is typically substantially homogeneous.On base material, the thickness of speckle is about 10 μm to 100 μ m.On base material, the thickness of each speckle is often substantially homogeneous, and the change between speckle to the thickness of speckle generally should minimize (for example, about 30 μm).

Target position

Used in literary composition, term " target position " refers to specific position on base material, material (such as host material, host material with add Plus agent, or analyte) can deposit and be retained in this position.Base material may include one or more target position, they can randomly or With orderly array or other pattern arrangement.When for mass spectral analyses (such as maldi analysis), target position or there are deposition materials The size of gained target position preferably equal to or smaller than will focus on the size of the LASER SPECKLE that desorbing is caused on base material.Therefore, target Position can be well or depression, contact pin or the beadlet being for example arranged on solid carrier surface, or physical barriers, or they Combination, such as the beadlet on chip, the chip in well, etc..Target position can be placed on base material by physics mode, Ke Yishi It is carved on substrate surface, can be " tower " staying after certain position perimeter etch, or can be to be joined by physical-chemical Numerical example such as relative hydropathy, hydrophobicity or any other surface chemical property limits, and liquid is retained in or on which Position.

Platform

The method of the present invention and and compositionss can be used in combination with any ionization source, these ionization sources include atmospheric pressure chemical Ionization (APCI), chemi-ionization (CI), electron bombardment (EI), electron spray ionisation (ESI or ES), fast atom bombardment (FAB), field Desorbing/FI (FD/FI), substance assistant laser desorpted ionized (MALDI) and thermospray ionization (TSP).In some embodiment party In formula, ionization source is MALDI or ES.

The method of the present invention and compositionss can be used in combination with any mass-synchrometer.It is currently available that mass-synchrometer has Multiple, wherein relatively common be quadrupole flight time (TOF) analyser, sectorial magnetic field, and Fourier transform and quadrupole from Sub- trap.In addition, this analyser can be used in series, become series connection (tandem as tandem) (MS-MS) mass spectrograph.At some In embodiment, mass-synchrometer is TOF analyser.

In some embodiments, by mass spectral analyses, analyte is analyzed, and without analysis of spectral method analyte. In some embodiments, analyte is not analyzed, the party by infrared spectrum (such as Fourier transform infrared spectroscopy) Method generally measures the frequency of vibration of some molecules, does not generally include the ionization of analyte and the survey of the analysis amount of substance to ionization Amount.

Embodiment

Following examples are nonrestrictive, for some embodiments of the present invention are described.

Embodiment 1:The additive of analyte

In the examples below, will be from Sequenom iPLEX^TMThe analyte of reaction and nanopure water or ascorbic acid Additive mixes, to determine the impact that additive is formed to adduct.According to the Sequenom iPLEX described in documents below^TMRule Journey abreast processes each block of plate：Jurinke,C.,Oeth,P.,van den Boom,D.,MALDI-TOF mass spectrometry:A versatile tool for high-performance DNA analysis (MALDI-TOF matter Spectrum：A kind of multifunctional efficient DNA analysis instrument), Mol.Biotechnol.26,147-164 (2004)；Oeth, P. etc., iPLEX^TMAssay:Increased Plexing Efficiency and Flexibility for System through single base primer extension with mass-modified Terminators (iPLEX^TMTest：Improved by single base primer extension and modification of liquor quality terminatorThe clump effect of system Rate and motility), SEQUENOM Application Note (2005), two documents are all incorporated herein by reference.Dilute Release/nurse one's health in step, according to indicated by standard schedule, 9 mouthfuls of l analyte solutions are mixed with 25 mouthfuls of l nanopure waters, also will simultaneously Other plates are mixed with ascorbic acid, and producing final ascorbic acid concentrations is 20mM.

In order to remove in ascorbic acid remaining cation, and the cation exchange resin of exclusion ammonification may bring Any interference, carries out desalinization process with the protonation resin Ascorbic Acid stock solution of 1g/ml.According to workflow, add Plus agent can directly be mixed with analyte, or dilute further, be subsequently adding in sample.In this particular embodiment, dilution step Suddenly complete on automatic liquid body processor, dilute solution volume ratio deposit with 1/25 before being added to analyte solution exists In 50ml disk.

After each dilution/conditioning step, using Sequenom nanometer allotter, two boards are all distributed with 10 nanoliters/region To pre- matrixing standard SpectroChip^TMOn (300mM 3-HPA/25mMDAC), in Sequenom It is analyzed on Analyzer Compact.

Result：Increase by 8% using the calls that ascorbic acid leads to the sample that Ascorbic Acid processed.In ascorbic acid In the sample processing, sodium and ammonia adduct are suppressed to Monitoring lower-cut.So lead to the sample that Ascorbic Acid was processed not There is the false positive calling leading to due to ammonia and/or sodium adduct, and the ammonia adduct in the sample of water conditioning leads to once survey Allozygote (false positive calling) is pointed in examination repeatedly.

Embodiment 2:The additive of substrate

In the examples below, the new base composition that display comprises ascorbic acid (AA) can reduce adduct and be formed, Thus improving spectrogram quality.

Matrix solution

(stock solution of different substrates component is according to component to prepare substrate combination by following stock solution and nanopure water Functional group's cation exchange resin carries out processing the acid with exchanger resin of-H+ form, NH4+ form with resin Ammonia salt)：

3-HPA:350mM, in 30% acetonitrile solution.

AA:The aqueous solution of 1M.

DAC:226mg 1Min aqueous solution.

Prepare standard substrates with 300mM 3HPA and 25mM DAC, and use 300mM 3HPA, 20mM NH₄- oxalates and 20mM ascorbic acid prepares new substrate.Referring to table 1.Using Gesim Nanoplotter, final substrate is distributed with 15-20nl To on SpectroChip.

Table 1

Synthetic oligonucleotide sample

Tested twice using synthetic oligonucleotide in table 1 substrate for the direct point sample.In the first experiment, " old " all test 17 mer synthetic oligo (GTG in resin processed substrate and the modified substrate of " new " ascorbic acid GTG GTG GTG GTG GT).In Fig. 1 (old substrate), hence it is evident that there is ammonia adduct, (peak heights are about the mother at 5335Da The 12% of peak heights), and in Fig. 2, there is no ammonia adduct.New substrate is also referred to as " substrate 63C ".

In second experiment, test low quality 17 mer synthetic oligo (5044Da) and high-quality 28 aggressiveness closes The adduct becoming oligonucleotide (8436Da) is formed and depurination.In testing twice, using Gesim Nanoplotter Synthetic oligonucleotide analyte is assigned in the substrate of table 1 with 10nl/ speckle.Low quality oligonucleotide and high-quality oligonucleoside The result of acid is respectively displayed in table 2 and table 3.

Table 2A：17 aggressiveness adducts

Table 2B:17 mer Probe height and signal to noise ratio (SNR)

Table 3A:28 aggressiveness adducts

Table 3B:28 mer Probe height and signal to noise ratio (SNR)

Probe
				Probe height (mean/standard deviation)	20/5	28/7	31/7
Probe SNR (mean/standard deviation)	93/19	134/27	164/31

Substrate is relatively based on adduct and depurination peak heights relatively, and probe height and SNR.In analysis Introduce adduct to form fraction the relative frequency of the speckle (pad) exceeding peak fraction threshold values to be described：

Adduct formation fraction=

=(exceeding the relative frequency of the speckle of threshold values) * (average adduct peak heights)

" speckle " and " speckle " on term base material is used interchangeably.The standard deviation of average adduct peak heights is relatively low, All suitable for all substrate, it is not reported in table.

Compared with through the standard substrates of resin treatment, for synthesizing 17 aggressiveness, the average adduct shape in new substrate Component number little by 13% (table 2A), for synthesizing 28 aggressiveness, the average adduct in new substrate forms fraction little by 29% (table 3A).

Primer extension sample

Carry out extra experiment using the effective Sequenom Genotypic tests pointing to RhD and AMG gene polynorphisms. These tests use iPLEX^TMTest andTechnology carries out (Jurinke, C., Oeth, P., van den Boom,D.,MALDI-TOF mass spectrometry:a versatile tool for high-performance DNA Analysis (MALDI-TOF mass spectrum：A kind of omnipotent high-performance DNA analysis instrument), Mol.Biotechnol.26,147-164 (2004)；Oeth, P. etc., iPLEX^TMAssay:Increased Plexing Efficiency and Flexibility forSystem through single base primer extension with mass-modified Terminators(iPLEX^TMTest：The mass spectral analyses being improved by single base primer extension and modification of liquor quality terminatorSystem Clump efficiency and motility), SEQUENOM Application Note (2005), this two documents are all by reference to being incorporated into This).In brief, expanded with PCR first around the target region of SNP.Subsequently, oligonucleotide primers are annealed with PCR primer, and Extended by single nucleotide acid allele-specific with the mixture of 4 kinds of terminator nucleotides and archaeal dna polymerase.Produce extending Thing is transferred on the chip array of miniaturization, is analyzed with MALDI-TOF mass spectrum.The determination of the molecular weight of extension products is permissible Positively identify SNP allele present in sample.Equipotential in given sample can be predicted by the peak area ratio of quality signal The relative abundance of gene.

Here experiment in, to point sample in table 1 shown in substrate on 7316Da AMG primer extension product use high by 20% Laser energy (with above-mentioned synthetic oligonucleotide experiment compared with).As shown in table 4 and 5, compared with standard substrates, distribution is in new base The average adduct of the extension products in matter forms fraction and reduces (reducing 40-50%), and the laser energy of increase is to adduct shape Become no to negatively affect.

Table 4A：AMG tests (7316Da) adduct standard laser energy

Table 4B:AMG test (7316Da) highly with signal to noise ratio (SNR)

Table 5:AMG tests the laser energy that (7316Da) adduct increases

In order to test the effectiveness of new substrate in the presence of compared with multiple analyte, in the substrate of table 1, amount of analysis increases (10,15 and 20nl) 7528Da AMG primer extension product.Referring to table 6 below.It is 10 and 15nl stylish substrate in analyte volume Average adduct to form fraction relatively low；And in 20nl, identical fraction (0.6) is observed to two kinds of substrate.

Table 6:In AMG test (7528Da) adduct substrate, analyte sample increases

Improved mass spectrum

Fig. 3 and Fig. 4 shows the result of HD-4-psi 3-i Genotypic tests, thus clearly illustrates minimizing adduct shape Eliminate the importance of false positive calling in pairs.It is not the insertion peak that standard substrates are reported at the peak of 7626Da (insertion) (Fig. 3), the but (NH of the extension products peak 55Da at 7571Da₃+ K) adduct peak.In new substrate This adduct peak clearly eliminates (Fig. 4).

Embodiment 3:Substrate and the additive of analyte

It is also tested for the modified substrate of new ascorbic acid (to be located at combining of the ascorbic acid being added to analyte solution The AMG primer extension product of 8289Da).As shown in the table 2-6 of embodiment 1 and embodiment 2, it is added in analyte or and standard The ascorbic acid of substrate combination considerably reduces ammonia and sodium adduct.Referring to table 7 below.Processed with ascorbic acid on standard substrates For the analyte crossed, slip reaches 43%, for distribution is in the acid-treated analyte of the use Vitamin C in new substrate, Slip reaches 57% (with distributing compared with the undressed analyte in undressed substrate).

Table 7:It is added to the ascorbic acid of analyte and substrate

Embodiment 4:Stability test

Within the time of 6 months, carried out four groups of stability tests, with determine ascorbic acid reduce adduct property with The change of time.There is no indication that tested substrate shows hydraulic performance decline within this time.On the contrary, obtained SNR and the adduct stable to ammonia and alkaline adduct form fraction and confirm 3-HPA and its additive DAC, NH₄- oxalic acid The stability combining of salt and ascorbic acid.

The full content of each patent, patent application, publication and the document herein quoted all is incorporated by reference into This.The quoting of above-mentioned patent, patent application, publication and document is not offered as applicant and recognizes that any of the above described content is related Prior art, is also not offered as recognizing the interior perhaps date of these publications or document.

In the case of without departing substantially from basic sides of the present invention, the above can be changed.Although with reference to one or Multiple specific embodiments describe the present invention in detail, but it will be appreciated by those of ordinary skill in the art that can have in the application The embodiment that body discloses is changed, as long as these changes and improvements are within the scope and spirit of.

Herein the present invention of suitably description can be implemented in the presence of any element no specifically disclosing herein.Cause This, for example, in each example of this paper, term "comprising", " substantially by ... form " and " by ... form " in any One can be replaced with any one in other two.The term having used and expression are as illustrative and not limiting property Term, the use of these terms and expression be not precluded from shown and described feature or part thereof any equivalent feature or Its part, and the various feasible changes in the scope of the invention requiring patent right.Term " one " or " a kind of " modification During certain element, (for example " device " can represent one for expression one or this element a kind of or this element multiple or multiple Or multiple device), unless be apparent that described is a kind of element or more than one element by context.Wen Zhong The term " about " being used represents the value (i.e. ± 10%) in the range of the 10% of underlying parameter, uses at the beginning of a train value Term " about " represents that each value in this train value (that is, " about 1,2 and 3 " are about 1, about 2 and about 3) modified in this term.For example, " about 100 grams " weight may include the weight between 90 grams to 110 grams.Although thus, it will be appreciated that the present invention is implemented by representativeness Mode and optional feature have been described in detail, but those skilled in the art are it is conceivable that the changing of the disclosure herein Become and variant, these change and variant is considered as falling within the scope of the present invention.

Set forth embodiments of the present invention in the dependent claims.

Claims (23)

1. a kind of host material for matrix-assisted laser desorption/MALDI-MS, it comprises：

Reduce the additive of adduct, this additive bag contains ascorbic acid or its salt, tautomer or the like；And

The compound being selected from the group for one or more：3- hydroxy-picolinic acid (3-HPA), dibasic ammonium citrate (DAC), 2,5- dihydroxy Yl benzoic acid (DHB), alpha-cyano -4- hydroxycinnamic acid (CHCA), 2,4,6- trihydroxy-acetophenone (THAP), T-2- (3- (uncle 4- Butyl phenyl) -2- methyl -2- allylidene) Cyanoacetyl-Cyacetazid (DCTB), dithranol (DIT), sinapic acid (SA), trans -3- indole third Olefin(e) acid (IAA), 2- (4- hydroxyphenyl azo) benzoic acid (HABA)；

The analog of wherein said ascorbic acid comprises following formula: compound：

Wherein R¹And R²It is independently OH, halogen, R³、OR³, azido, cyano group, CH₂R³、CHR³R⁴、SR³Or NR³R⁴；

R³And R⁴It is independently H, alkyl, acetenyl or cyano group, or optionally substituted aryl carbocyclic ring, aryl-heterocyclic, non-aryl carbocyclic ring Or nonarylheterocyclic.

2. host material as claimed in claim 1, the content range wherein reducing the additive of adduct is to account for host material About 99 weight % of gross weight are to about 1 weight %.

3. host material as claimed in claim 1, the additive bag wherein reducing adduct contains ascorbic acid.

4. host material as claimed in claim 1, wherein said compound comprises 3- hydroxy-picolinic acid (3-HPA).

5. the host material as any one of claim 1-4, wherein said compound comprises dibasic ammonium citrate (DAC).

6. host material as claimed in claim 4, also comprises ammonium oxalate.

7. the host material as any one of claim 1-4 and 6, the concentration of the additive of described minimizing adduct is about 5-50mM.

8. the host material as any one of claim 1-4 and 6, the concentration of described compound is about 1mg/ml-20mg/ ml.

9. host material as claimed in claim 7, the concentration of described compound is about 1mg/ml-20mg/ml.

10. a kind of base material, it comprises carrier, and described carrier includes array of spots, and wherein one or more described speckles comprise to weigh Profit requires the host material described in 1.

11. base materials as claimed in claim 10 are it is characterised in that the additive bag reducing adduct contains ascorbic acid.

12. base materials as claimed in claim 10 are it is characterised in that described substrate comprises 3- hydroxy-picolinic acid (3-HPA).

13. base materials as claimed in claim 12 are it is characterised in that described host material also comprises ammonium oxalate.

14. base materials as claimed in claim 13 are it is characterised in that the concentration of the additive of described minimizing adduct is about 5- 50mM.

15. base materials as claimed in claim 13 are it is characterised in that also comprise the ascorbic acid 3- pyridone first of about 300mM Sour (3-HPA).

16. base materials as claimed in claim 15 are it is characterised in that also comprise the ascorbic acid of about 20mM.

17. base materials as claimed in claim 16 are it is characterised in that also comprise the ammonium oxalate of about 20mM.

18. base materials as claimed in claim 10 are it is characterised in that described one or more speckle includes analyte.

19. base materials as claimed in claim 18 are it is characterised in that described analyte is nucleic acid.

A kind of 20. methods preparing the base material being suitable for matrix-assisted laser desorption/MALDI-MS, the method includes：

So that the amount of adduct formation can be reduced under Mass Spectrometry Conditions in deposited on substrates host material as claimed in claim 1；

Wherein said host material is deposited on base material in the way of array of spots, and

The described additive bag reducing adduct contains ascorbic acid or its salt, tautomer or the like.

21. methods as claimed in claim 20 are it is characterised in that described base material includes Silicon stone.

22. methods as described in claim 20 or 21 are it is characterised in that each speckle on array comprises (i) 3- pyridone Formic acid (3-HPA) and (ii) ascorbic acid or its salt, tautomer or the like.

23. methods as claimed in claim 22 are it is characterised in that each speckle on array also comprises ammonium oxalate.